For successful gene transfer to primitive hematopoietic cells several requirements need to be achieved. These include identification of the desired target cell population, identification of the appropriate vector to be used, and achieving desired levels of gene expression. To date, successful gene transfer in human subjects remain problematic. To address these problems as well as important safety issues, studies in non-human primates are performed in this program to optimize gene transfer to nonhuman primate hematopoietic cells prior to human clinical studies. Vectors that were evaluated over the past year were constructed to express the enhanced green fluorescent protein (EGFP) reporter gene, the neomycin resistance gene, the common gamma-chain cytokine receptor, the multiple drug resistance gene, and a variant of the dihydrofolate resistance gene. Transduction conditions employed the RGD-containing fibronectin (FN) fragment, RetroNectin (CH-296) and a variety of recombinant hematopoietic growth factors which included stem cell factor, interleukin-6, megakaryocyte growth and differentiation factor (MGDF or thrombopoietin) and the human Flt-3 (fms-like tyrosine kinase) ligand. Retroviral vectors included a murine stem cell virus(MSCV)-based vector, Moloney murine leukemia (MLV)-based vectors, and a third generation human immunodeficiency virus type-1 (HIV-1) lentiviral vector. MSCV-based vectors were pseudotyped with the envelope protein of an endogenous xenotropic feline virus, RD114 or the gibbon ape leukemia virus, PG13, and the third generation lentiviral vector was pseudotyped with the vesicular stomatitis virus G-protein. Our efforts over the past year have involved identifying transduction protocols for both murine retroviral vectors and human lentiviral vectors which optimized prolonged high-level detection of retrovirally marked hematopoietic cells in nonhuman primates following transduction of CD34+ cells. Methods employing various cytokine combinations and serum-free conditions using the FN fragment CH-296 have proven effective. Persistent multiple lineage marking using clinically feasible protocols ranged from 5-15%, with transient marking as high as 70%. In addition, these studies demonstrated prolonged expression of reporter genes, such as EGFP, and preliminary studies are under way evaluating functional gene expression looking at drug resistance. Successful long-term expression of foreign genes, such as the jellyfish protein EGFP or the bacterial neomycin resistance gene appears to be associated with tolerance induction associated with transplant radiation and assays are being developed to determine the extent of this tolerance. Despite these successes, questions remain. How can consistent high levels of expression be obtained? Are true hematopoietic stem cells being targeted? Can other stem cells either derived from bone marrow or other easily accessible tissues be targeted to assist in either the contribution or repair of other organs? These questions are being addressed currently through the evaluation of new vectors as they are being developed, further delineation of the nature and clonality of the population contributing to the reconstitution, and with the initiation of new studies to isolate, characterize, and transduce rare populations of cells that may contribute to organogenesis.